Download Practical aspects of Microscopy

Practical aspects of Microscopy
1
Basic
Conventional Optical Microscopy
Scanning Electron Microscopy
Related techniques
Some slides borrowed from Prof. Ashish
Garg’s lecture on “Structure and
Characterization of Materials”
Knowledge Incubation for TEQIP IIT Kanpur
2
Microscopy Techniques
Microscopy is a field of investigation which is used to study objects which are
too small to be easily viewed by the human eye. Viewing and studying
objects that range in size from millimeters (1 mm = 10-3 meter) to
nanometers (1 nm = 10-9 meter) intrigues everyone and is currently
applied to every field of science and technology in use today. Microscopes,
devices which magnify, come in a wide range of forms and use a multitude of
illumination sources ( light, electrons, ions, x-rays and and mechanical
probes) and signals to produce an image. A microscope can be as simple
as a hand held magnifying glass or as complex as a multi-million
dollar research instrument. Using these tools, a microscopist explores
the relationship of structure and properties of a wide variety of
materials in order to better understand the reasons why a particular item
behaves the way it does.
Knowledge Incubation for TEQIP IIT Kanpur
Microscopy Techniques
3
Microscopy can be broadly subdivided into two
categories
Optical microscopy
Conventional light microscopy, Fluorescence microscopy,
confocal/multiphoton microscopy and Stimulated
emission depletion microscopy
Electron probe microscopy
Scanning electron microscopy (SEM), Transmission
electron microscopy (TEM), Scanning transmission
electron microscopy (STEM), Focus ion beam microscopy
(FIB)
Knowledge Incubation for TEQIP IIT Kanpur
Optical Microscope
4
Simple Light Microscope
Reflected Light Microscope
Contrast
Modes of Analysis
Bright Field/ Dark Field/ Phase Field/ Polarized Light
Knowledge Incubation for TEQIP IIT Kanpur
Conventional Optical Microscopy
5
This is an optical instrument containing one or more lenses that produce
an enlarged image of an object placed in focal plane of the lens
Magnification is given by M= v/u
1. Transmission: beam of light passes through the sample
2. Reflection: beam of light reflected off the sample surface
e.g. Metallurgical or reflected light microscope to study surface of opaque
materials
Knowledge Incubation for TEQIP IIT Kanpur
6
A simple light microscope
The eyepiece is placed such that the image formed by the objective falls at
first focal point of the eyepiece. The light thus emerges as parallel rays.
The distance between the focal plane of the objective (f1) and the focal plane of the
eyepiece (f2) is called the tube length (l). The object to be viewed is placed just
outside the focal point at the left side of the objective lens. The enlarged image is
formed at a distance l + f1 from the objective.
Knowledge Incubation for TEQIP IIT Kanpur
An Optical Microscope (Reflected Light)
7
The illuminating system
Source of light illuminating
the sample
The specimen stage
holds the sample in position
and controls the x, y and z
coordinates of the area
under observation
The imaging system
Transfers a magnified and
undistorted image to the
plane of observation and to
the recording medium.
Knowledge Incubation for TEQIP IIT Kanpur
Applications of Optical Microscopy
Optical micrographs of 1040 steel after polishing with a sequence of diamond grits:
(a) Rough-grinding to achieve a planar surface; (b) after polishing with 6 mm
diamond grit; (c) after polishing with 1 mm diamond grit; (d) after polishing with 1/4
mm diamond grit.
Knowledge Incubation for TEQIP IIT Kanpur
Applications of Optical Microscopy
The same 1040 steel after etching for
different lengths of time in a very dilute
nitric acid: (a) under – etched; (b) a good
etch; (c) over etched
Knowledge Incubation for TEQIP IIT Kanpur
Metallography guidelines
10
If a mounted specimen is used, an adherent mount is
very important, else ‘bleeding’ can occur
The sample must be free of scratches, disturbed metal
and any kind of embedded contaminants.
The specimen must be thoroughly cleaned before
etching.
After etching, the sample should be rinsed in HOT water,
followed by an alcohol rinse and dried under HOT air.
If additional etching time is required, the specimen
should be re-rubbed a few seconds on a final polishing
wheel
https://www.cartech.com/techarticles.aspx?id=1450
Knowledge Incubation for TEQIP IIT Kanpur
11
camera
Beam
splitter
Reflected light
Transmitted light
Knowledge Incubation for TEQIP IIT Kanpur
Olympus
BX51
Research
Microscope
Cutaway
Diagram
Contrast
Contrast is defined as the difference in light intensity between the
specimen and the adjacent background relative to the overall background
intensity.
Image contrast, C is defined by
Ssample − Sbackground ∆S
C=
=
Ssample
SA
Ssample and Sbackgroud are intensities
measured from specimen and
background, e.g., A and B, in the
scanned area.
Knowledge Incubation for TEQIP IIT Kanpur
12
13
Angle of Illumination
Bright filed illumination – The normal method of illumination, light comes from
above (for reflected OM)
Oblique illumination – light is not projected along the optical axis of the objective
lens; better contrast for detail features
Dark field illumination – The light is projected onto specimen surface through a
special mirror block and attachment in the objective – the most effective way to
improve contrast.
Light stop
Imin
Imax
C=
Imax-Imin
Imax
C-contrast
Knowledge Incubation for TEQIP IIT Kanpur
Bright Field Imaging
14
• Simplest and most common
• White light and contrast in the sample is
caused by absorbance of some of the
transmitted/reflected light
Advantages
• Simplicity of setup with only basic
equipment required.
Stainess Steel 330
Limitations
• Very low contrast of most biological
samples.
• Low apparent optical resolution due to
the blur of out of focus material.
Source: Wikipedia
Knowledge Incubation for TEQIP IIT Kanpur
Dark field Imaging
15
An illumination technique used to
enhance the contrast
Illuminates the sample with light
that will not be collected by the
objective lens, and thus will not
form part of the image.
This produces the classic
appearance of a dark, almost
black, background with bright
objects on it.
Knowledge Incubation for TEQIP IIT Kanpur
16
Contrasting examples of dark field (left) versus bright field (right)
illumination of SS330. Dark field shows grain boundaries while bright field shows
grain structure
See more at:
http://www.microscope-detective.com/dark-field-microscope.html#sthash.95GiEgYC.dpuf
Knowledge Incubation for TEQIP IIT Kanpur
17
Polarized light microscopy is utilized to
distinguish between singly refracting
(optically isotropic) and doubly
refracting (optically anisotropic) media
Knowledge Incubation for TEQIP IIT Kanpur
18
Polarized Light Microscope Configuration
Knowledge Incubation for TEQIP IIT Kanpur
Phase Contrast Microscopy
Conversion of phase shifts (typically invisible) in light that passes through a
transparent specimen to brightness changes in the image which are now visible.
Source: Wikipedia
Knowledge Incubation for TEQIP IIT Kanpur
19
Phase Contrast Microscopy
20
Phase contrast is based on the destructive interference of
light scattered from small features associated with a
difference in height h.
Based on the idea that the scattered amplitude is
approximately π/2 out-of-phase with the specularly reflected
beams.
A “phase shift ring” ensures that the background light is also
put out-of-phase by π/2
“Bumps” and “hollows” in the surface topology will appear
either brighter or darker than the background in the image
because of interference with background light
Colorless and transparent specimen, such as living cells and
micro-organisms
Knowledge Incubation for TEQIP IIT Kanpur
21
Example (a cell)
• Useful in biology
bright field image
phase contrast image
Source: Wikipedia
Knowledge Incubation for TEQIP IIT Kanpur
Limitations: Resolution
22
DIFFRACTION AND RESOLUTION LIMIT
(http://hyperphysics.phyastr.gsu.edu/hbase/phyopt/mulslid.html#c2)
Knowledge Incubation for TEQIP IIT Kanpur
Knowledge Incubation for TEQIP IIT Kanpur
Numerical Aperture (NA)
24
Angular aperture
(≤72 degrees)
Even an aperture in front of
the lens can change the
value of NA
α
One half of A-A
NA of an lens is a measure of its ability to gather light and
resolve fine specimen detail at a fixed object distance.
NA = n(sin α)
n: refractive index of the imaging medium between the front lens of
objective and specimen cover glass
Dr. Shashank Shekhar
Materials Characterization
Knowledge Incubation for TEQIP IIT Kanpur
Limitations: Depth of Field
25
Knowledge Incubation for TEQIP IIT Kanpur
26
n : index of
refraction of the
medium between
the objective and
the object.
So smaller NA is
better for depth-offield, but what are we
losing?
Knowledge Incubation for TEQIP IIT Kanpur
27
If Disc of confusion is smaller than resolution,
then does it matter that the focus is not proper?
Knowledge Incubation for TEQIP IIT Kanpur
28
Depth of Field
Since the resolution is finite, the object need not be in the exact object plane in
order to remain in focus: there is an allowed depth of field d. Similarly, the image
may be observed without loss of resolution if the image plane is slightly displaced:
there is an allowed depth of focus D.
d=
nλ  n 
+
e
2
NA  M .NA 
First term is Diffraction limited d-o-f
λ: Wavelength of illumination
Second term is geometry related d-o-f
n: Refractive index of the imaging medium
NA: Objective numerical aperture
M: Objective lateral magnification,
e: Smallest distance that can be resolved by a detector that is placed in the image plane of the
objective.
Knowledge Incubation for TEQIP IIT Kanpur
(d-o-f) Optical Microscopy vs
Scanning Electron Microscopy
29
25µm
radiolarian
OM
Small depth of field
Low resolution
SEM
Large depth of field
High resolution
J.I. Goldstein et al., eds., Scanning Electron Microscopy and X-Ray Microanalysis,
(Plenum Press,NY,1980).
Knowledge Incubation for TEQIP IIT Kanpur
30
Scanning Electron Microscope
1938 ( Von Ardenne)
http://www.ammrf.org.au/mys
cope/sem/
Principles of Electron
Microscopy
Knowledge Incubation for TEQIP IIT Kanpur
Electron Microscopy
31
Electrons from a source interact with
electrons in specimen yielding a variety of
photons and electrons via elastic and
inelastic scattering processes.
These are the “signals” that are used to
make images and measure or characterize
the structure composition of our
specimens
Knowledge Incubation for TEQIP IIT Kanpur
SEM: Why electrons?
32
(1) Wavelength: (Wave nature of Electron)
h
λ=
=
mv
h
1 + eV
2m0 eV (
)
2
2m0 c
≈ (1.5 / V )1/ 2 nm
• At 10 kV, typical of many applications of SEM, the wavelength is
only 0.012 nm, appreciably less than the interatomic distances in
solids.
Knowledge Incubation for TEQIP IIT Kanpur
SEM: Why electrons?
33
(2) Charge: (Particle nature of Electron)
Being charged particles, electrons can be deflected in
an electromagnetic-field and thus can be brought
to focus to very small region over the sample.
Compared to it, the photons in a beam of light
(electromagnetic waves) being neutral can not be
focused in this fashion.
Knowledge Incubation for TEQIP IIT Kanpur
34
SEM contains
broadly
Gun
Column of lenses
Specimen chamber
SE detector
Control Console
Knowledge Incubation for TEQIP IIT Kanpur
Beam – Specimen Interaction
35
Knowledge Incubation for TEQIP IIT Kanpur
Beam-Specimen Interaction
36
Elastic (a): incident electron’s
direction altered by Coulombic field
of nucleus. Direction may be
changed by 0-180° (ave 2-5°) but
velocity remains virtually constant.
<1 % of beam energy transferred.
Inelastic (b): incident electron
E0 = accelerating voltage (of
electrons emitted from gun);
usually 15-20 KeV for SEM
transfers some energy (up to all, E0)
to tightly bound inner-shell electrons
and loosely bound outer-shell
electrons.
(Goldstein et al, 1992, p.72)
Knowledge Incubation for TEQIP IIT Kanpur
Elastic and inelastic scattering
37
Slide from University of Tennesse
Knowledge Incubation for TEQIP IIT Kanpur
Ranges and interaction volumes
38
http://www4.nau.edu/microanalysis/Microprobe/Interact-Effects.html
Knowledge Incubation for TEQIP IIT Kanpur
Secondary Electrons
39
Produced by inelastic interactions of high energy electrons
with electrons of atoms in the specimen which cause the
ejection of the electrons from the atoms
After undergoing additional scattering events while
travelling through the specimen, some of these ejected
electrons emerge from the surface of the specimen.
Knowledge Incubation for TEQIP IIT Kanpur
Backscattered Electrons
40
• High energy beam electrons may suffer single or multiple elastic scattering
events in the solid, escaping from the material.
• The fraction of beam electrons that scatter back was found experimentally to
vary directly as a function of composition (atomic number Z).
• This provides a valuable imaging tool: a rapid means to discriminate phases
that have different mean Z values.
http://bioweb.usu.edu/emlab/TEMSEM%20Teaching/SEM%20backscattered.html
Intensity (grey level) varies from black
(voids/epoxy), to plagioclase, olivine, basaltic glass,
with Ti-magnetite the brightest phase.
Knowledge Incubation for TEQIP IIT Kanpur
Backscattered Electrons
41
Knowledge Incubation for TEQIP IIT Kanpur
Topographic Contrast
42
Image from Characteriation Facility Manual, University of Minnosota
Knowledge Incubation for TEQIP IIT Kanpur
Other Signals in SEM
43
Knowledge Incubation for TEQIP IIT Kanpur
Other Signals in SEM: X-ray (for EDS and WDS) and Auger
Electrons
44
Time
1
K shell
L shell
(=photoelectron
)
• HV electron knocks inner
shell (K here) electron out of
its orbit
• An electron from a higher
energy orbital (L here) ‘falls
in’ to fill the void (time=2).
2
Blue Lines indicate
subsequent times: 1
to 2, then 3 where
there are 2 alternate
outcomes
• Excess of energy released
as a photon
3
EDS/ WDS
• The photon can exit, either
by ejecting another outer
shell electron as an Auger
electron, or as X-ray
AES
(Goldstein et al, 1992, p 120)
Knowledge Incubation for TEQIP IIT Kanpur
45
Elemental
distribution over a
surface can be
obtained EDS
Knowledge Incubation for TEQIP IIT Kanpur
Electrons and X-rays …
don’t get them
confused !
46
• X-rays have no mass, no charge; electrons
have charge (key!) and a small mass
• X-rays can be produced both by accelerating
HV electrons -- and with other X-rays -- in a
vacuum and colliding them with a target.
• The resulting X-ray spectrum contains (1)
continuum or continuous background
(Bremsstrahlung), (2) occurrence of sharp
lines (characteristic X-rays), and (3) a cutoff
of continuum at a short wavelength/high
keV.
Knowledge Incubation for TEQIP IIT Kanpur